Cruise Report
CK09-01 Chikyu training cruise Leg. 1
Kumano Mud-Volcano Drilling:
A Window to the Deep-Biosphere
Mar. 8-18, 2009
JAMSTEC
1
Shipboard scientific party
Expedition Project Managers (EPMs): Nobu Eguchi (EPM-chief), Kan Aoike,
Yusuke Kubo, and Sean Taczko
Program Operation Group,
Center for Deep Earth Exploration (CDEX), JAMSTEC
Chief scientist: Fumio Inagaki (geomicrobiologist)
Geomicrobiology Group,
Kochi Institute for Core Sample Research, JAMSTEC
e-mail: [email protected]
Juichiro Ashi (geologist)
Ocean Research Institute,
The University of Tokyo, Japan
e-mail: [email protected]
Akira Ijiri (geochemist)
Institute for Research on Earth Evolution (IFREE),
Research Program for Paleoenvironment, JAMSTEC
e-mail: [email protected]
Hiroyuki Imachi (microbiologist)
Subground Animalcule Retrieval (SUGAR) Program,
Extremo-biosphere Research Center (XBR), JAMSTEC
e-mail: [email protected]
Yuki Morono (microbiologist)
Geomicrobiology Group,
Kochi Institute for Core Sample Research, JAMSTEC
e-mail: [email protected]
Ko-ichi Nakamura (geologist)
2
National Institute of Advanced Industrial Science and Technology (AIST)
e-mail: [email protected]
Takeshi Terada (microbiologist)
Marine Works Japan, Co., Kochi, Japan
e-mail: [email protected]
Tomohiro Toki (geochemist)
Department of Chemistry, Biology, and Marine Science,
Faculty of Science, University of the Ryukyus, Japan
e-mail: [email protected]
Yasuhiko Yamaguchi (biogeochemist)
Ocean Research Institute,
The University of Tokyo, Japan
e-mail: [email protected]
JAMSTEC Core Curator: Hideaki Matchiyama (sedimentrogist)
Kochi Institute for Core Sample Research, JAMSTEC
e-mail: [email protected]
3
Acknowledgement
The science party of the CK09-01 Leg 1 (Kumano mud volcano) wishes to thank
all crews of the deep-earth research drilling vessel “Chikyu” and drilling staffs of
Mantle Quest Japan (MQJ), Co., for providing an opportunity to study the
subsurface of the Kumano mud-volcano no. 5 during the CK09-01 Chikyu
training cruise. We are also grateful to the technical and curational staffs of
Marine Work Japan (MWJ) for their supporting efforts on the core processing
and laboratory experiments.
4
Introduction
The marine subsurface sediments harbor the largest microbial biomass
that comprises one-tenth of all living biota on Earth (Whitman et al., 1999). The
microbial population generally decreases with increasing burial depth because
of the limitation of bio-available nutrient and energy substrates as well as the
compaction of habitable space with depth burial (Parkes et al., 1994, 2000). The
metabolic activity depends on the flux of nutrient and energy mainly from either
the overlying seawater or the underlying basaltic aquifer (D’Hondt et al., 2004);
hence, the primary photosynthetic production in the water column and the fluid
flow regimes play significant roles in the nutrient and energy transport, affecting
the population, activity and diversity of sub-seafloor microbial community. The
recent biogeochemical studies demonstrated that the heterotrophic archaeal
cells dominate the sub-seafloor microbial community, which abundance shows a
parallel trend with organic contents in sediments (Biddle et al., 2006; Lipp et al.,
2008). The sub-seafloor microbial community is generally composed of
phylogenetically
diverse,
yet-to-be-characterized
archaeal
and
bacterial
components, whose genetic and physiological characteristics are largely
unknown, and hence their ecological and biogeochemical roles remain elusive
(Inagaki et al., 2003, 2006; Inagaki and Nakagawa 2008).
One of the key challenges in the scientific drilling is to comprehend the
extent of life and biosphere (D’Hondt et al., 2007). Recent study in the Ocean
Drilling Program Leg 210 off the Newfoundland margin showed that microbial
populations over 105 cells/ml are present down to 1626 meters below the
seafloor (mbsf) sediments that are 111 My old and 60º to 100ºC (Roussel et al.,
2008). However, it needs a repeated clarification by the third party because the
cell enumeration was performed by the manual count based on the conventional
direct eye-discrimination of acridine orange (AO) stained-cells, which often
causes serious overestimates of cell number because of the high fluorescent
backgrounds. Recently, the improved cell detection and enumeration technique
using computer-image calculation has been established, providing more precise,
objective, and hence reliable data of cell abundance in cored samples (Morono
et al., 2009). The new cell-enumeration technique is expected to be highly useful
5
for the future explorations of the deep sub-seafloor biosphere, including this
CK09-01 cruise.
During the CK09-01 Leg 1 Chikyu training cruise, we tested to drill a
mud volcano no. 5 in the Kumano forearc basin of the Nankai Trough, Japan.
This provides us an unprecedented opportunity to expand our knowledge on the
previously inaccessible deep and hot sub-seafloor biosphere because the mud
is originally derived from the deep realm down to a few thousands of meters
below the seafloor. The mud volcanoes are widely distributed in the prelate
convergent continental margins (Milkov 2000; Kopf 2002). The formation
process of mud-volcano is so called “diapirism”, in which a lower density, high
fluid content, deformable deep material intrudes upward into the stratified
overlying sediments along the deeply rooted fault since the density contrast acts
as a force of buoyancy. The diapiric mud typically contains consolidated to
semi-consolidated breccias trapped in the mud matrix during the upwelling
intrusion. The mud fluids typically have low chloride (Cl) and high boron (B) and
lithium
(Li)
concentrations
(Hensen
et
al.,
2007),
indicating
the
depth-pressurized dehydration of clay minerals (smectite-illite reaction) at high
temperature around 100ºC. The deep-sourced dehydrated water can be
distinguished from the seawater by deuterium (δD) and oxygen (δ18O) isotopic
compositions of the pore water. One of the other characteristics of the mud
diapir is the presence of a lot of hydrocarbon gasses including methane,
sometimes oil materials, produced by either or both microbial and/or
thermogenic processes (see a review of Etiope et al., 2009).
Although piston or gravity coring down to a few meters below the
surface of mud volcano has been demonstrated many times in multiple locations,
the deep scientific drilling has only been performed in the Ocean Drilling
Program (ODP) Leg 160 at Site 970, the East Mediterranean Sea off Italy. At
Site 970 Hole D, approximately 45.3 m cores were successfully retrieved using
an advance piston core (APC) barrel from the summit of the mud-volcano. The
pore water chemical analyses showed very low chloride concentrations (e.g., 61
mM at Core 4H-5, 30.7 mbsf), indicating two possibilities: 1) methane hydrate
dissolution or 2) mineral dehydration upon increasing pressure (De Lange and
Brumsack, 1998). The δD-δ18O isotopic study of the pore water conclusively
6
suggested that the fluids are originally derived from clay mineral dehydration
(mainly smectite-illite transformation), corresponding to a depth range of 3.5-7
km and an elevated temperature of about 120-160 ºC (Dählmann and De Lange,
2003). The deeply rooted hydrothermal imprint of clay-dehydrated low salinity
water has also been supported by Li-B enrichment (Hensen et al., 2007),
providing us an important implication to the occurrence of the deep, hot
biosphere that can be seen through the diapiric mudflow.
In general, the activity of sub-seafloor microbes on the continental
margin sediments largely depends on the organic matter buried from the
overlying seawater column; however, the young diapiric mud volcano, which
formed at an episodic geologic event such as faulting, has not been thickly
covered by fleshly buried sediments; hence, the ecosystem in the deep mud
volcano is expected to be different to the surrounding stratifying sedimentary
environment and to still maintain the deep sub-seafloor microbial ecosystem,
even in the relatively shallow part of the mound. In the Kumano basin of the
Nankai Trough, ten or bit more diapiric mound-like structures have been
observed by the seismic and sea-bottom surveys, among which the no. 5 mud
volcano is thought to form in relatively young period because of the absence or
very little coverage of overlying sediments. Furthermore, the bottom survey
using the manned submersible Shinkai 6500 revealed the presence of
chemosynthetic Calyptogena colony along the rim of the crater, indicating the
active seepage of hydrocarbons along the outer surface of the intrusion
pathway.
The surface sediments of mud-volcano have been well studied where
the microbes strongly mediate an anaerobic oxidation of methane (AOM) at
methane seep sites of the volcanic summit. For example, at the Haakon Mosby
mud volcano in the Barents Sea, anaerobic methanotrophic archaea (ANME-2 &
3) and sulfate reducing bacteria (Desulfosarcina, Desulfococcus, Desulfobulbus)
consume <40% of the total flux of methane in the surface sediments close to the
sulfate-methane interface (Niemann et al., 2006). Geochemical characteristics in
the surface sediments of the Haakon Mosby mud volcano were studied down to
approximately 300cm below the seafloor (Ginsburg et al., 1999). However, the
microbiological and biogeochemical characteristics in deep realms have
7
remained elusive.
Methane hydrates were sometimes observed in or near the mud
volcano. For example, the presence of methane hydrates associated with mud
volcano was reported from the Barbados accrationary wedge (Martin et al.,
1996), and the Middle American Trench, offshore the Costa Rica (Milkov, 2000)
with gravity and/or piston coring. Massive hydrates were retrieved from
2m-gravity core sediment of the mud diapir no. 11 on off Costa Rica (Schmidt et
al., 2005). Hensen et al. studied δD and δ18O isotopic compositions of pore water
from the hydrate-bearing mud samples and estimated the temperature range of
the clay dehydration from 85 ºC to 130 ºC (Hensen et al., 2004). Methane
hydrates were also reported from the mud volcano in the Gulf of Gaidz, in which
pore waters contained low chloride concentrations potentially caused by the
hydrate formation (Mazurenko et al., 2003). The presence of methane hydrates
was widely observed in the Kumano forearc basin in the Nankai Trough. The
seismic survey around the mud volcano no. 5 showed the presence of a
bottom-simulating reflector (BSR) at around 340 mbsf in the flank sediments;
however, no BSR or hydrate-indications have been observed in the body of mud
volcano.
Despite the importance of diapiric environment as “A Window to the
Deep-Biosphere”, there is no demonstration to study microbiology and
biogeochemistry in the deep realms of the mud diapir, probably because of the
limited opportunity to obtain the drilled core samples. During the CK09-01
Chikyu training cruise (Leg. 1), we have an opportunity to study the diapiric
materials down to approximately 20 mbsf from the mud-diapir center (Site
C9004) and the crater rim (Site C9005) close to Calyptogena colonies.
Our major scientific goals during this exploration are:
1) to understand the carbon and nitrogen biogeochemical cycles,
2) to understand the microbial population, community structure and
diversity,
3) to understand the microbial metabolic rates and networks,
4) to retrieve the geochemical, biological, geological signatures reflecting
the deep, high temperature zone close to the mud-diapiric origin,
8
5) to understand the formation process of the mud-volcano no. 5, and
6) to discuss the habitability of life in the deep realm associated with
dehydration process.
To accomplish these scientific objectives, we perform multidisciplinary study
including geology, organic and inorganic geochemistry, microbiology, and
molecular biology using the cored sediments, and try to comprehend the unique
environment as a window to the deep sub-seafloor biosphere.
Core Handling Flow and Site Summary
Location map of the mud volcanoes in the Kumano basin of the Nankai Trough.
9
Core handling flow
Since the sediment core contains a lot of breccias with mud matrix, the vertical
core splitting into working half and archive half is expected to be difficult to
perform during the limited shipboard time. It may need a special tool or
technique to make an undisturbed cutting surface, and time-consuming, thus we
decided not to split core during the expedition time, instead, will transport the
whole round core to the Kochi Core Center for the post-cruise processing.
During the CK09-01 Leg 1 cruise at the Kumano basin, we tested coring with
hydraulic piston coring system (HPCS). Since the surface sediments might
contain high concentration of hydrogen sulfide (H2S), we put cascade musk for
processing safely on the core cutting area and rig floor, and released
high-pressure gas by making small hales on the plastic liner to prevent core
expansion. After core recovery on the cutting area, we immediately monitored
temperature
anomalies
caused
by
methane
hydrate-dissolution
using
thermo-view IR camera, resulting in the successful identification of the location
of methane hydrates. After core sectioning and curational work on the core
cutting area, we took samples directory from core-section ends for the
headspace gas measurement (903HS), microscopic work (903FISH), and
moisture and density measurement (903MUD). Then, we took XCT images of all
the core sections and checked lithological features of the non-destructive WRCs,
providing useful information for the subsequent WRC processing for
geochemistry and microbiology. The WRC samples were collected in the QA/QC
lab. During the WRC cutting, all cores were stored at reefer room to keep the
cool temperature for living materials (i.e., microbial activities). The WRC
samples were then distributed in geochemists and microbiologists for further
sub-sampling steps and onboard analyses. The cores were then subjected to
non-destructive routine measurements using MSCL-W (multi-sensor core
logger), and finally stored at 4 ºC reefer. The cores will be sent to Kochi Core
Center for further processing, MSCL measurements of the split cores, and
sub-sampling for the additional geochemical and geological studies.
10
11
Core processing on the core cutting area. All scientists and MWJ technicians working on the
core cutting area put an air cascade on face because of the production of toxic hydrogen sulfide
(H2S) from the core materials.
Site C0004
Site C0004 is located in the diapiric crater center of the mud-volcano no. 5. The
water depth was 1896.7 m, and the site position was 136º 34.0851'E, 33º
40.5486'N. We started drilling on March 08 and recovered sediments down to
19.7 mbsf (Hole A, Core 1H-3H) by using hydraulic piston coring system (HPCS)
during one-day coring operation. Since the production of H2S from the surface
sediments was expected, all the processing on the cutting area was performed
with putting on the air-cascade musk. The core sediments were composed
mainly of clay mud including consolidated to semi-consolidated sedimentary
breccias. The core contained a lot of hydrocarbon gases; hence the cores were
generally expanded with many small voids and some sediment portions were
blowout from the core liner during the recovery. The temperature monitoring
using a thermo-view IR camera revealed the presence of methane hydrates in
several horizons at Site C0004. Several whitey massive particles of methane
hydrates as well as a short WRC section were immediately stored in PFA cups
with liquid nitrogen on the core cutting area, and processed to onboard
geochemical analyses. The pore water chemical and dissolved gas analyses
12
revealed that chloride concentrations of the massive methane hydrates were
extremely low (~4.7 mM) and C1/C2 values were notably higher than those in
mud matrix sediments (<200 mM: see Geochemistry). The sediments were
mostly sulfidic with low sulfate concentrations, indicating the occurrence of
microbial anaerobic oxidation of methane (AOM) coupled to strong sulfate
reduction according to the following equation: CH4 + SO42- → HCO3- + HS- +
H2O. Preliminary data of δD and δ18O isotopic compositions of pore water (H2O),
which were measured onboard by an infrared isotopic mass spectrometry,
indicated that, although the hydrate formation may somewhat disproportionate
the isotopic values, the fluids are originally derived from the deep realm via the
pressure-dehydrated clay minerals.
Site C0005
Site C0005 is located in the southwestern rim of the mud-volcano where the
chemosynthetic Calyptogena colonies are spotty inhabiting sulfidic surface
sediments associated with methane seepage. First, we tried to make Hole A
close to a big Calyptogena colony at the water depth of 1886.7m (33º 40.5579' N,
136º 33.9396' E). However, the core barrel was broken presumably by the pipe
swelling, thus the sediment recovery was unfortunately null. Then, we moved a
few meters northeast and made Hole B at the water depth of 1887.7 m (33º
40.5615' N, 136º 33.9401' E), where several Calyptogena mussels dispersedly
inhabited. Although we could have successfully recovered the surface core from
Hole B, some upper portions of the surface sediments was unexpectedly
blowout from the corer on the rig floor, potentially because of the dissolution of
massive methane hydrates during the core recovery and pipe handling.
Approximately 6m-sediment core was left on the core liner; however, the
sediments were appeared to be terribly destructed by the gas explosion. Then,
we slightly moved east and made Hole C again (1887.7 m in water depth, 33º
40.5615' N, 136º 33.9406' E), and retrieved the cores at almost the same point
down to 13.2 mbsf using a combination of drilling and HPCS coring. The cored
sediments were overall gassy with methane. We observed visible massive
methane hydrates in the sediment matrix down to 12.7 mbsf (Hole D, Core 1H-5).
The thermo-view IR camera revealed that the entire sediments in Hole D,
13
especially in Core 1H-4 and 5, widely contained methane hydrates. The two
sections, 1H-4 and 1H5 in the depth range from 11.8 to 12.8 mbsf, were
immediately transferred to the XCT-room and scanned the images before the
hydrates melt. As far as we know, this is probably the first direct XCT analysis of
natural hydrates in the mud diapir and other environments. The sediment cores
down to 12 mbsf produced a lot of H2S as the sensor always indicated red,
suggesting the active microbial sulfate reduction coupled with methane (i.e.,
AOM) in the hydrate-bearing muddy sediments. Several pieces of massive
hydrates and several hydrate-bearing sediment samples were quickly frozen by
liquid nitrogen on the core cutting area, and preserved in PFA cup at -150 ºC for
the subsequent geochemical and microbiological analyses. Interestingly, the
core sediments below 13 mbsf from Hole E did not contain methane hydrates
and sulfide. The XCT-scanned image showed a mass gradient structure of
breccias, indicating the overflowing mud with breccias during the diapir formation
process, which is a different sedimentological feature to the diapiric center at
Site C0004 (see Geology section). The sulfate concentrations in the upper
sedimentary unit above 13mbsf were very low or absent while the Hole E
sediments contained over 25 mM, which is similar to the seawater. (see
Geochemistry section) These results indicate that the outcrop of the mound top
is highly permeable because of the mud-matrix-poor breccias, and hence
secondary seawater circulation occurs, which may continuously supply oxidants
(electron acceptors) such as oxygen or sulfate to microbes inhabiting the
adjacent muddy environments associated with methane hydrates.
14
Thermo-view IR camera monitoring of negative temperature anomaly caused by the dissolution
of methane hydrates. The photos show Core 1H-5 at Site C9005, Hole D, in which visible whitey
massive hydrates were widely distributed.
15
Microbiology and Biogeochemistry
During the CK09-01 Leg 1 cruise at the Kumano mud volcano, we have
processed core samples to store under appropriate conditions for shore-based
microbiological and biogeochemical analyses. The quick sample processing is
extremely important because most microbes in marine subsurface are most
likely very sensitive to oxygen and high temperature, and once the microbes are
dead the fragile bio-molecules such as RNA or intact polar lipids may be
enzymatically degraded. Except for 903FISH and 903GH samples collected on
the cutting area, all samples were collected as whole round core (WRC) in the
QA/QC lab on the Chikyu, immediately after the XCT scanning. On behalf of the
scientific party, the chief scientist made quick decisions for the WRC sampling
location according to the core recovery, sampling depth interval, and lithology
based on the XCT-scanned image. The sub-sampling procedure and the
shore-based experimental plans for each sample are described as follows;
Sample Code: 903FISH. For microscopic works including cell enumeration and
fluorescent in situ hybridization (FISH), 5 cm3 of the innermost-core sediments
was collected by a tip-cut sterilized syringe from all core section-ends and
903ACT samples. The sediments from the core section-end were collected
immediately after the core splitting on the cutting area. The FISH samples from
903ACT were obtained in a lamina-flow clean bench of microbiology lab. 1 cm3
of sediment was mixed with 9ml of 4% paraformaldehyde in phosphate buffer
saline (PBS: pH 7.6), vortexed vigorously, and then incubated for 6-8 hours at
4ºC. If there are some consolidated sediment particles during the vortex mixing,
we used an ethanol-wiped spatula for breaking the structure for the complete
slurry. For 903FISH slurry, we used 15 ml round-end plastic centrifuge tube for
easy mixing. After the fixation, the sediment slurry was centrifuged at 3,500xg
for 10 min at 4ºC, discarded the supernatant, and added 9ml PBS for washing
sediments (i.e, removal of excess paraformaldehyde). The sediments were
voltexed again, and centrifuged at 3,500xg for 10 min at 4ºC. We performed this
washing step twice. The final volume was adjusted at 10ml with PBS:ethanol
solution (1:1), vortexed, and then stored at -20ºC. The sediment residue
16
(approximately 4 cm3) was separately put into 15 ml plastic tube and stored at
-80 ºC as the reference.
Sample Code: 903ACT. To study microbial metabolic activities, 20 cm WRC
samples were collected in the QA/QC lab. 1 cm3 of the sediments was first taken
for microscopic works, and fixed as described above. The rest sediments were
replaced into Ar-flushed autoclaved 1L glass bottle with ethanol-wiped spatulas.
After the replacement, we additionally flushed the bottle with Ar for a few
minutes to completely remove oxygen from the bottle. The reason why we use
Ar for flushing instead of nitrogen is to prevent microbial nitrogen assimilation,
which might be critical for subsequent isotope-labeled experiments. The sample
bottle was sealed with a black butyl rubber stopper and a screw cap, and then
stored at 4 ºC.
As for the shore-based experiments to measure the potential activity rates, we
will perform some incubation experiments using 14C and 35S-labeled radiotracers
for organic-consuming sulfate reduction rate, methane-consuming sulfate
reduction
rate,
homo(CO2-reducing)-methanogenesis
rate,
aceticlasitc
methanogenesis rate, homo-acetogenesis rate, and acetate-oxidation rate. After
the incubations with radiotracers, metabolic products such as sulfide, methane,
and acetate will be extracted from the incubation slurry and the radioactivity will
be measured by scintillation counter. We also study the potential assimilation
activities using stable isotope (13C and 15N)-labeled substrates such as methane,
formate, acetate, bicarbonate, glucose, pyruvate, ammonia, nitrate, nitrite, and
amino acids for carbon and nitrogen sources. All the incubation experiments for
the metabolic activities will be conducted using 903ACT sediment slurries,
supplemented with anaerobic artificial water close to in situ salinity and pH
conditions. After the incubations, substrate incorporation rates, population ratios,
and the phylogenetic identifications will be studied using NanoSIMS (nano-scale
secondary ion mass spectrometry) at single cell level as well as at biomarker
level.
Sample Code: 903CULT. For the cultivation experiments, 5 cm to 10 cm WRC
samples were collected in the QA/QC lab, immediately transferred to a clean
17
bench, and then the innermost-core sediments were put into autoclaved 250 ml
glass bottle with continuous supply of nitrogen. The bottle was sealed with butyl
rubber stopper, and then stored at 4 ºC. The trimmings of core surface were
separately put into a zip-loc vinyl bag, and stored at -80 ºC, which will be used
for the extraction of co-nutrient sources from the mud.
Cultivation provides direct evidences for the existence of active microbial
components and sometimes results in isolation of new species, hence
microbiologically very important. Previous cultivation studies of sub-seafloor
microbes have suggested that the predominant microbial components, most of
which are phylogenetically distinct from any previously known isolates, are
highly resistant to the conventional batch-type cultivation technique or need long
time for the growth. Using the 903CULT samples from the mud-volcano no. 5,
we try to make enrichment cultures by using a flow-through-type reactor system
and to reproduce ex situ acetate/bicarbonate-based microbial ecosystem. In
addition, we try to cultivate thermophilic to hyperthermophilic microbes including
archaea from the mud diapir samples, since the diapiric mud is derived from
deep, hot sub-seafloor environments.
Sample Code: 903MBIO. For molecular (DNA and RNA) and biomarker
analyses of the microbial community in the mud samples, 10 cm to 15 cm WRCs
were collected in the QA/QC lab. The WRC was put into a zip-loc vinyl bag, and
immediately stored at -80 ºC. For the better preservation of RNA, 50 cm3 of
innermost-core sediments were taken by an ethanol-wiped spatula and
autoclaved 50 ml tip-cut syringe, put the sediment into an autoclaved 125 cc
PFA (perfluoroalcoxyalkane) cup, added 50 ml RNA later solution (Ambion), and
then stored at -80 ºC. The frozen WRC samples will be aseptically cut without
melting using a newly developed electric scope saw system at JAMSTEC-Kochi
for sharing the samples at exactly the same horizon between molecular and
biomarker experiments.
Our recent study of extracted DNA and intact polar lipids (IPLs) suggested that
the sub-seafloor stratified sediments harbor a large population of heterotrophic
18
Archaea (Lipp et al., 2008). However, the microbial community structure and its
metabolic functioning in the mud diapiric habitat from shallow to deep have
remained completely unknown. Using 903MBIO samples, we extract bulk
DNA/RNA and IPLs and quantify the amount of archaeal and bacterial 16S
rRNA gene copy numbers and IPLs. The extracted DNA/RNA will be amplified
with phi29 polymerase with careful contamination monitoring with real-time PCR.
Using amplified environmental genomes, the copy numbers of some key
functional genes will be surveyed (Inagaki et al., 2004): e.g., methyl-co-enzyme
M reductase (mcrA) gene for methanogens and methanotrophs, dissimilatory
sulfite reductase (dsrAB) gene and dissimilatory adenosine-5’-phsphosulfate
rudcatase (apsA) gene for sulfate reducers, and formyl tetrahydrofolate
synthetase (fhs) gene for acetyl-coA pathway. The genetic information will be
useful to construct the potential metabolic network and ecosystem in the mud
diapiric environment. If RNA is successfully extracted from metabolically active
microbial assemblages, we try to study for the first time the functioning structure
of whole amplified messenger-RNA (so called “metabolome” analysis) in
sub-seafloor environment, if possible.
Sample Code: 903BAC. A few samples of 10cm WRC were collected in the
QA/QC lab for the purpose of “bio-archive”. To test the better preservation of
RNA, we used 903BAC sample from the top 10 cm of Core 1H-1 at Site 9004
Hole A, and stored 25cm3 surface sediments at different conditions as follows: 1)
store at 4ºC, 2) store at -20 ºC, 3) store at -80 ºC, 4) store at -80 ºC with an equal
volume of RNA later, 5) store at -20 ºC with an equal volume of acetone, 6)
snap-freezing in liquid nitrogen (-196 ºC) and store at -80 ºC, and 7)
snap-freezing in microwave freezer and stored at -80 ºC. The several other
903BAC samples were stored at -80 ºC in zip-loc vinyl bag. The bio-archive
samples will be aseptically sub-sampled with an electric scope saw, and stored
in liquid nitrogen tank (-160 ºC) according to the policy of JAMSTEC core
curation.
Sample Code: 903GH. Because the existence of methane hydrates was
expected based on the physical and geochemical condition of the mud-volcano
19
no. 5, we monitored the temperature anomalies caused by the dissolution of
hydrates with Thermo-View IR camera on the cutting area as described above.
We found over 20 horizons of the lower temperature anomalies at both Sites
C0004 and C0005, indicating the widespread existence of methane hydrates in
the mud volcano no. 5. We collected several hydrate horizons as WRC, put the
hydrate-bearing sediment into the autoclaved PFA (perfluoroalcoxyalkane) cup,
and then pored liquid nitrogen directory on the samples on the cutting area. The
frozen hydrate samples were then stored at -150 ºC, which will be used for
geochemical and molecular biological analyses.
Cell Count
903FISH
(1 cc)
Ethanol:PBS (1:1)
up to 10 cc
Fixation (4% PFA) centrifuge
Wash twice with PBS
3500xg,10min
4ºC, 6-8 hours
-20ºC
FISH
Flow Through Ractor
903CULT
5cm WRC
(175 cc)
250ml bottle
with N2 flush
Trimming
(150 cc)
4ºC
High Pressure Bach Cultivation
Radioactive Tracer Experiments
Trimming
(600 cc)
903ACT
20cm WRC
(700 cc)
1000ml bottle
with Ar flush
4ºC
Bio-marker Isotope
Stable Isotope Probing
FISH (1 cc)
NanoSIMS
-80ºC
add 50 cc RNA later
RNA (50 cc)
903MBIO
10cm WRC
(350 cc)
-80ºC
DNA/IPL (300 cc)
903BAC
10cm WRC
(350 cc)
PFA cup
liquid N2
quick frozen
-150ºC
KCC
Liquid N2 tank
molecular analyes
903GH
10cm WRC
(350 cc)
liquid N2
quick frozen
-150ºC
XCT image scan
KCC
Liquid N2 tank
geochemical analyses
A summary flow chart of the sample processing and the shore-based analyses for microbiology
and biogeochemistry.
20
Geochemistry
A total of 20 and 4 WRC samples (903IW) were collected in the QA/QC lab for
onboard interstitial water analyses at Sites 9004 and 9005, respectively. After
the XCT-scan and WRC sampling, interstitial waters were squeezed from WRCs
(10 to 55 cm in core length), and salinity, pH, alkalinity, chlorinity, Br, SO4, Na, K,
Mg, Ca, Sr, Ba, Mn, Li, B, Mn, Fe, Si, Sr, Ba, NH4, V, Cu, Zn, Rb, Mo, Cs, Pb,
and U concentrations as well as hydrogen and oxygen isotopic composition of
water were analyzed onboard. Fore shore-based analyses such as iodine
analyses, ~18.1 ml water sample was sub-sampled from each IW sample and
stored under the appropriate conditions.
For the analyses of hydrocarbon and hydrogen gases, a total of 18 and 14
sediment samples (3 cm3) were collected by tip-cut sterilized syringes at Sites
9004 and 9005, respectively, immediately after the core recovery on the core
cutting area. Approximately 3 cm3 sediment was placed in a 50 cm3 glass vial
with a degassed HgCl2 solution, and then the vial was sealed with a Teflon
coating butyl septum and a metal crimp cap.
At both Site C0004 and C0005, the cores contained gas hydrates at several
horizons. 2 and 3 massive whitey gas hydrate samples (ca. 5-cm3) stored in
liquid nitrogen were used for chemical analyses at Sites 9004 and 9005,
respectively. We thawed the hydrate sample in a 1000 cm3 glass bottle filled with
nitrogen inside and sealed with a butyl rubber septum. Then, the melted water
and released gases in the bottle were extracted by a pipette and a gas-tight
syringe, respectively.
Explanatory Notes for Onboard Chemical Analyses on Chikyu
Salinity was determined by a reflect meter. pH was determined by an electrode.
Alkalinity and chlorinity were determined according to the routine titrimetric
methods in the IODP. Br, SO4, Na, K, Mg, and Ca were determined by an ion
chromatography. Sr, Ba, Mn, Li, B, Mn, Fe, Si, Sr, and Ba were determined by
21
ICP-AES. V, Cu, Zn, Rb, Mo, Cs, and Pb were determined by ICP-MS.
Hydrogen and oxygen isotopic compositions of water were determined by a
laser
(infrared)
adsorption
spectroscopy
(Los
Gatos
Research,
Inc.).
Concentration of hydrocarbon and hydrogen gases was determined by GC-FID
and GC-TRD, respectively.
The sub-sample processing and storage conditions for the onshore analyses
were as follows:
1) Hydrogen isotopic composition of H2 (Subsample Code: 903IWH); as a
pretreatment, HgCl2 regent was supplemented into 20-cm3 glass vial that was
sealed with a Teflon-coated butyl septum and metal crimp cap. Then, 3ml
water sample was placed into the vial via the septum by using a plastic
syringe. The vial was stored at -20 ˚C.
2) Concentration of trace gases (CH3Cl, COS, etc) (Subsample Code:
903IWTG); HgCl2 regent was supplemented into 20-cm3 glass vial that was
sealed with a butyl septum and metal cap. Then, 3 ml water sample was
placed into the vial via the septum by using a plastic syringe. The vial was
stored at -20 ˚C.
3) Concentrations and δ13C of CO2 and CH4 (Subsample Code: 903IWC1); 2 ml
water sample was placed in 2-cm3 glass vial with amidosulfuric acid and
HgCl2. The vial was sealed with a butyl septum and metal crimp cap and
stored at room temperature.
4) Concentrations and δ13C of DOC and acetate (Subsample Code: 903IWC2); 4
ml water sample was placed into 10-cm3 screw top glass vial. The glass vial
was stored at -20 ˚C.
5) Concentration of iodine (Subsample Code: 903IWI1); 1 ml water sample was
placed into 4 ml high-density polyethylene screw top vial. The vial was stored
at 5˚C.
6) Isotopic composition of iodine (Subsample Code: 903IWI2); 5 ml water
sample was placed into 8 ml high-density polyethylene screw top vial. The vial
was stored at 5
˚C.
22
Preliminary Results: Interstitial Water and Gas Hydrate
Site 9004
One of the unique characteristics of interstitial water in the mud volcano’s core is
its significantly low chlorinity relative to seawater (Fig. Geochemi-1a). The
chlorinity values were ranging from 242 mM at 0.7 mbsf as maximum to 150 mM
at 3 mbsf. Bellow the depth of 3 mbsf, the chlorinity showed little change, and
averaged 130 mM. This value is 23% of chlorinity in seawater. Preliminary
results of the stable isotopic compositions of the interstitial water showed
18
O-enriched and D-depleted values relative to those in seawater, indicating that
the most fluids were derived from the dehydrate reaction of clay minerals in
deeply buried marine sediments. The high concentrations of B (~13 mM) were
observed in 903IW samples, consistently indicating that the fluids were derived
from clay mineral dehydration at a temperature range from 60˚C to 150˚C
(Haese et al., 2006). The SO4 concentrations were overall very low (<1.5 mM),
even in the shallowest depth of 0.7 mbsf, and decreasing in parallel with depth
down to 3 mbsf, 0.5 mM (Fig. Geochemi-1b). The overall low concentrations of
SO4 indicate the occurrence of microbial sulfate reduction in the cored samples.
Chlorinity of the formation water of massive gas hydrate samples (903GH)
was found to be extremely low as the minimum value at 4.7 mM (Fig.
Geochemi-1a). The stable isotopic compositions of the water exhibited both 18Oand D-enriched values, indicating that the gas hydrates are composed of the
deeply rooted interstitial water in the sediment matrix because it is known that
gas hydrates preferentially incorporate the heavier oxygen and hydrogen
isotopes as its formation water (Kvenvolden and Kastner; 1990; Martin et al,
1996; Matsumoto and Browski, 2000).
Site 9005
The chemical characteristics of the interstitial waters at Site C0005 were
identified as low chlorinity, high δ18O and low δD, and high B contents, which
were similar to those at Site C0004 and consistently indicated that the most
fluids in the southeastern rim of the mud diapiric crater were derived from the
deep sedimentary column. The chlorinity of the shallowest 903IW sample was
23
382 mM and fell down to 170 mM at 9.15 mbsf of Hole D (Fig. Geochemi-1a), in
which the existence of gas hydrates was observed by the visual inspection and
the Thermo-View IR camera. In clear contrast to the upper hydrate-bearing mud
layers, the deeper core section at 12.92mbsf, Hole E (1H-1) contained high
chloride (537mM) and SO4 (28.8mM) concentrations in the interstitial water
close to those in seawater (Fig. Geochemi-1b). The cored materials in Hole E
were mainly composed of silty grains and breccias, hence the seawater may
infiltrate into this sedimentary unit through the large pore spaces.
(a)
(b)
Fig. Geochemi-1. Vertical profile of (a) chlorinity and (b) SO4 concentrations at Sites 9004 and
9005.
Preliminary Results: Gas compositions
Concentrations of CH4, C2H6, H2, and CO in the gas-phase of sediment samples
(903HS) and gases from dissociated gas hydrate samples (903GH) were
measured in the geochemistry lab of Chikyu. The presence of ethane and the
low to intermediate C1/C2 ratios of 130 to 1500 for all the samples (Fig.
Geochemi-2) suggests that the gasses at Sites C0004 and C0005 are the
mixture of microbial and thermogenic origins. Noteworthy, a very low C1/C2 ratio
less than 100 was observed in the gas hydrate sample at Site C0004, indicating
a strong contribution of thermogenic gas in the hydrate sample; however, it is
24
unknown why such extremely low C1/C2 ratio occurs in the hydrate sample
specifically and what is the effect of microbial contribution (i.e., ethanogenesis)
on the gas composition associated with the hydrate sample. Conclusively, the
results of gas-analysis indicate that the pore fluids are generally enriched in
thermogenic gas transported from greater depths with some contributions of
localized microbial activities in shallow habitats.
Fig. Geochemi-2. Vertical profiles of CH4 to C2H6 (C1/C2) molecular ratios in headspace gas
(903HS) at Site C0004 and C0005. Trends with depth for C1/C2 ratios at Site C0004 and Site
C0005 are indicated by circler and triangle plots, respectively. X- and Y-axis represents C1/C2
ratio and sediment depth, respectively. The data for the gas from dissociated gas hydrate
samples (903GH) are also plotted in the profile by open circles and open triangles for Site C0004
and Site C0005, respectively.
Shore-based geochemical study
The results from onboard analyses indicated that most fluids were derived from
the dehydrate reaction of clay minerals in deeply buried marine sediments. It
highly supports our expectation that the mud volcano is
“A Window to the
Deep-Biosphere”. To better understand the biogeochemical processes in the
deep biosphere, we plan to do isotopic analyses for various materials such as
acetate, CH4, and ΣCO2 etc., which play important roles for biogeochemical
25
carbon cycling naturally occurring in the Kumano mud volcano no. 5. In 2006, we
retrieved a short piston core (1.5 m length) from around summit of the
mud-volcano no. 5 during KH06-03 cruise of R/V Hakuho in 2006. The carbon
isotopic compositions of acetate and DIC, DOC, and methane showed that the
δ13C values of acetate increased with increasing depth (from -41 to -21‰).
Interestingly, the δ13C values of ΣCO2 consistently increased from -19 to +33‰.
The parallel correlation of δ13C values between acetate and ΣCO2 is most likely
caused by the acetogenesis, in which anaerobic bacteria (i.e., homo-acetogens)
produce acetate via the reductive CO2-pathway (i.e., acetyl-coA pathway). The
extremely heavier shift of δ13C of ΣCO2 may indicate in situ consumption of CO2
by autotrophic microbes such as methanogens and homo-acetogens; however,
the CO2-consuming microbiological characteristics have remained elusive.
Since the 1.5 m-core recovered during the KH06-03 cruise was too short for the
deep-biosphere research, the CK09-01 Leg 1 coring expedition provides us an
unprecedented opportunity to study the continuing isotopic trends of key
substrates for the carbon biogeochemical cycles in the deeper area of the mud
volcano no. 5, which is our on going experimental effort as the shore-based
geochemical experiments.
26
Geology
Moisture and Density Measurements (MAD) and Whole Round
Multi-Scan Core Logger (MSCL-W) Measurements
Method: MAD measurement
Index properties of sediments were determined by measuring wet mass, dry
mass, and dry volume. The method was followed by the standard procedure of
the IODP MAD measurement (see Method chapter of IODP Proceedings 315).
Approximately 5 cc of samples were taken for MAD from each section bottom.
Sediment samples for headspace gas analysis were also obtained from the
same locations. The water content data measured by MAD were shared with
geochemists for the headspace gas measurement.
Index property measurements were conducted on 32 samples. After
measurement of wet sediment mass, dry sediment mass and volume were
measured after drying in a convection oven for 24 hours at a temperature of 105
± 5 °C. Wet and dry masses were weighed using paired electronic balances and
dry
volume
was
measured
using
a
helium-displacement
pycnometer
(Quantachrome Penta-Pycnometer). A constant 1.024 g/cm3 was used on
calculation for pore fluid density.
Method: MSCL-W measurements
MSCL is composed of the following four logging systems for unsplit core:
Gamma Ray Attenuation (GRA) Density, Magnetic Susceptibility (MS), Natural
Gamma Radiation (NGR) and P-Wave Velocity (PWV) tools. These
measurements are conducted routinely as a standard measurement item for
IODP. The method used on this study is followed by the standard procedure of
the IODP MSCL-W measurement (see Method chapter of IODP Proceedings
315).
Method: Thermal Conductivity measurement
All recovered cores from mud volcanoes are highly disrupted due to expansion
of gassy sediments. We abandoned thermal conductivity measurements
27
because a large number of tiny bubbles severely affect on thermal conductivity
values.
Results of MAD and MSCL-W measurements
Physical property of sediment is one of the important factors recording states of
compaction and digenesis. Mud volcanoes, targets of our study, are basically
formed by buoyancy of under-consolidated muddy sediment with reversed
density to the surrounding formation. Therefore, physical properties of erupted
sediments are significant for understanding formation processes of mud
volcanoes. We obtained whole round MSCL data and index properties of
discrete samples onboard.
Muddy matrix sediments were mainly collected from section ends using 12
ml syringe for MAD measurements although core samples include various sized
clasts. 18 samples were measured down to 20 mbsf at Site C9004. One sample
is exceptionally a semi-consolidated mud clast. At Site C9005, MAD data were
obtained on 14 samples. We observed a small piece of methane hydrate on one
sample at about 6.5 mbsf. The sample from the Hole E bottom at about 13 mbsf
is characterized by clast-bearing muddy sediment, Bulk density, porosity and
grain density from MAD measurements are shown in Figures MAD-1 and MAD-2.
These figures also show results of bulk density derived from gamma ray
attenuation and magnetic susceptibility by MSCL measurements.
Bulk density at Site C9004 shows almost constant values from the seafloor
to 20 mbsf (Figure MAD-1). Grain density also indicates no systematic trend with
depth. Variations of porosity basically well correspond to changes of bulk density.
The MAD data from a clast sample were characterized by high density and low
porosity. This suggests that the clast was derived from nearby deeper
semi-consolidated host-rock.
Bulk density and porosity at Site C9005 indicate constant values from the
seafloor to 9 mbsf (Figure MAD-2). Data from 9 to 13 mbsf show considerably
lower bulk density and higher porosity values than those from the shallower
sequence. Such soupy sediments indicate either the dissociation of methane
hydrate or the severe mixing with seawater by coring disturbance although
hydrate itself was not recognized in these MAD samples. Grain density indicates
28
no systematic trend with depth. Clast-bearing mud form the bottom was
characterized by high density and low porosity due to existence of consolidated
host-rock pieces.
Bulk density values estimated from MSCL at Sites C9004 and C9005 are
much lower than those from MAD measurements (Figures MAD-1 and MAD-2).
This discrepancy was caused by insufficient sediment fillings of core liners
because these data were calculated as full sediment fillings in core liners.
Magnetic susceptibility data from MSCL, which have similar patterns to bulk
density from MSCL, also didn’t exhibit correct changes with depth, but just filling
ratios in core liners. We need volume correction on MSCL data for further
discussion on bulk density and magnetic susceptibility.
Voids in core liners severely affect on P-wave velocity measurements on
MSCL. Heterogeneity of voids in core liner make impossible to obtain correct
electrical resistivity by MSCL. Therefore, we took no account of results from
these measurements.
In conclusion, there is no systematic trend on muddy matrix samples by
MAD measurements at both drilling sites. This result suggests the diapirism of
low-density mud and little modification by compaction after the diapirism. MAD
data of clasts are limited, but significant for estimating depth of host rock
formation. One of important shore-based studies is further MAD measurement of
clasts from split cores.
29
Fig. MAD-1. Bulk density, porosity and grain density from MAD measurement, and bulk density
derived from gamma ray attenuation and magnetic susceptibility by MSCL measurement at Site
C9004.
Fig. MAD-2. Bulk density, porosity and grain density from MAD measurement, and bulk density
derived from gamma ray attenuation and magnetic susceptibility by MSCL measurement at Site
C9005.
30
Visual and X-ray CT (CAT) scan geological observation
During the CK09-01 (Leg 1) cruise at the Kumano mud volcano no. 5, all of the
cores were scanned by the General Electric Healthcare Light Speed Ultra 16
X-ray computer tomographic scanner installed in the core laboratory of Chikyu.
The supplemented protocol no. 6.6 was used throughout the scanning works.
The main specifications of this protocol were as follows:
Scan type: Helical Full scan by 0.5 sec rotation time
Helical thickness: 0.625 mm, SFOV: small, DFOV: 9.6 cm
X-ray source tube voltage: 120 kV, current: 100 mA
We tentatively analyzed the XCT-scanned data using OsirX ver. 3.3.2 software
connected to the XCT-DICOM on the core processing deck of Chikyu.
Site C0004
The cores at Site C0004, the central part of summit crater of the Kumano mud
volcano no. 5, were composed of consolidated to semi-consolidated mud rock
fragments within the matrix mud. Several millimeter-sized spherical voids were
predominant throughout the core images, indicating the gas separation from
interstitial water at and near the surface. Thermo-View IR camera imaging of the
core liner surface on the core cutting area clearly indicated the presence of gas
hydrates, which are presumably composed of methane. The CT images showed
that the size and fragments of mud rock clasts become bigger as the depth
increased. Below the bottom of the core 2, the piston cored off some bigger mud
fragments, which filled the entire core in the section images. These are the
evidence that the expelled mud was composed of not only fluidal mud but also
harder fragments of mudstone, which were the main components of building
cone-shape structure under the water. Hence, it was assumed that the fluidal
mud could not produce convex upward structure on the seafloor without the
harder mud rock fragments.
Site C0005
The main constituents (i.e., mud rock fragments and matrix mud) of the cores at
Site C9005 were similar to those at the C9004, as well as spherical voids in
31
various degrees. The mud rock fragments at Site C9005 were relatively smaller
than those at Site C9004. Gas-hydrates were abundant at Site C0005, enabled
us to make a visual inspection of gas-hydrate texture on the CT images. In Core
1 of Hole E, two sequences of graded bedding of fine sand to cobble size mud
clasts and fragments were observed on the CT images. The upper sequence of
graded bedding contained some mollusk shell fragments. Both graded bedding
and shell fragments indicated that these units in the Hole E were reworked
sediments of chemosynthetic colonies on the expelled mud.
Because we did not cut the cores in half on board, the observation was limited
and was far from full lithological description of the cores, which will be performed
at the Kochi Core Center as the post-cruise study.
Additional remarks: The other drilled cores at Site NT2-11 (for Geotech study)
during the CK09-01 Chikyu training cruise have lots of small partings and cracks
in mud section at the interval of several mm to several cm along the cores, which
were most likely produced by piston coring. This low core quality hinders digital
data processing of CT images as well as most data analysis of physical property
measurements such as gamma attenuation and P-wave velocity. The core
quality should have to be improved for future science programs to maximize the
usefulness of high-tech and modern instrumental facilities of Chikyu for
advanced science.
32
CK09-01 Leg 1 Kumano Mud Volcano No. 5 Site C0004 XCT-Scan Image
(cm)
0
1H-1
1H-2
1H-3
1H-4
1H-5
1H-6
1H-cc
2H-1
2H-2
2H-3
2H-4
2H-5
2H-6
2H-7
2H-8
2H-cc
3H-1
3H-2
3H-3
3H-4
3H-5
3H-6
3H-cc
50
100
150
33
CK09-01 Leg 1 Kumano Mud Volcano No. 5 Site C0005 XCT-Scan Image
(cm)
1H-1
0
Hole C
Hole B
1H-2
1H-3
1H-4
1H-5
1H-6
1H-7
1H-8
1H-cc
1H-1
1H-2
1H-3
1H-4
Hole D
1H-5
1H-6
1H-cc
1H-1
1H-2
1H-3
1H-4
Hole E
1H-5
1H-cc
1H-1
1H-cc
50
100
150
34
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37
Appendix 1. Curational Section Summary
38
39
40
41
Interstitial Water
Activity
IW
ACT
MBIO
Cultivation
Molecular Biology
BAC
Bio-Archive Core
CULT
(cm)
1H-1
0
1H-2
CK09-01 Leg 1 Kumano Mud Volcano No. 5 Site C0004 XCT-Scan Image & Whole round Sample
1H-3
1H-4
1H-5
1H-6
1H-cc
2H-1
2H-2
2H-3
2H-4
2H-5
2H-6
2H-7
2H-8
2H-cc
3H-1
3H-3
3H-2
3H-4
3H-5
3H-6
3H-cc
MBIO
IW
ACT
MBIO
ACT
CULT
ACT
IW
IW
CULT
IW
IW
IW
IW
BAC
CULT
CULT
IW
BAC
MBIO
50
MBIO
MBIO
ACT
CULT
MBIO
IW
CULT
IW
CULT
IW
ACT
MBIO
IW
ACT
CULT
IW
100
CULT
ACT
MBIO
CULT
ACT
ACT
IW
IW
ACT
IW
IW
IW
MBIO
CULT
MBIO
BAC
MBIO
150
42
IW
ACT
CULT
MBIO
BAC
GH
(cm)
1H-1
0
Interstitial Water
Activity
Cultivation
Molecular Biology
Bio-Archive Core
Gas Hydrate
1H-2
1H-3
CK09-01 Leg 1 Kumano Mud Volcano No. 5 Site C0005 XCT-Scan Image & Whole round Sample
Hole C
Hole B
1H-4
1H-5
1H-6
1H-7
1H-8
1H-cc
1H-1
1H-2
1H-3
1H-4
Hole D
1H-5
1H-6
1H-cc
1H-1
1H-2
1H-3
Hole E
1H-4
1H-5
1H-cc
1H-1
1H-cc
GH
GH
MBIO
IW
IW
CULT
ACT
IW
ACT
50
CULT
MBIO
IW
CULT
100
150
43